The intracellular concentration of Hb S is a powerful determinant of the kinetic and equilibrium properties of Hb S polymerization and cell sickling. A possible therapeutic strategy for sickle cell disease is based on preventing the loss of K which leads to the characteristic dehydration of sickle erythrocytes (RBC) and increases cell Hb S concentration. Blockade of the Gardos channel with clotrimazole(CLT) reduces sickle cell dehydration in vitro and in vivo. There are no specific pharmacological inhibitors of the KC-I cotransport system, the other major determinant of sickle cell dehydration. However, it has been shown that intracellular Mg is a powerful modulator of K-CI cotransport. Since an increase in intracellular Mg inhibited sickle cell dehydration in vitro, we had proposed to study whether dietary Mg supplements could be beneficial in sickle cell disease. Our studies have shown that dietary Mg supplements reduce RBC dehydration in a transgenic mouse model for sickle cell disease (Blood 1996;88:2738) and in patients with sickle cell disease (JCI 1997;100:11847). Long-term use of Mg supplements also reduces the incidence of painful crises in SS patients (Blood, 1997; abstract in press). We propose to pursue this line of research with the following new specific aims: 1) Characterize the role of Mg in regulating ion transport and cell volume in normal and sickle erythrocytes: we will determine in normal and sickle (human and mouse) the molecular mechanisms underlying the effects of Mg on the K-Cl co-transporter (KCC1), focusing our efforts on the regulatory interactions between Mg and RBC protein phosphatases and kinases that modulate K-Cl cotransport. 2) Study the cellular effects of long-term Mg supplementation therapy in SS and SC disease and of combination therapy of Mg supplements with either clotrimazole or hydroxyurea: We will focus on the effects of Mg therapy on RBC K-Cl cotransport regulation, Na/Mg exchanger activity, Mg content in density separated cells, and morphologic sickling. Mg therapy will be assessed in SC disease, which shows prominent dehydration via K-Cl cotransport. Combination studies with CLT or hydroxyurea will follow in SS disease. (3) Assess the effects of different Mg preparations on the acute and chronic manifestations of the disease in a transgenic mouse model: We will determine the effect of long-term Mg supplementation using different Mg preparations on the response to acute and chronic hypoxia, on the pathological manifestations and life expectancy of a transgenic mouse model of sickle cell disease. These objectives are the logical extension of the data generated during the prior cycle of funding. They are consistent with the expertise of the applicants and aimed to develop a new therapy for sickle cell disease.